Development of mutations in DNA repair deficient stationary phase cells of Saccharomyces cerevisiae

Genome integrity is essential for the health of the individual. Mutations, which can occur in resting cells as well as in proliferating cells, may probably contribute to cancer and aging. Since a large fraction of human somatic cells are in a non-replicative state it is necessary to understand mechanisms in non-replicating cells. In this diploma thesis I observed the different aspects of the onset of mutations in replicating and non-replicating cells of Saccharomyces cerevisiae (S. cerevisiae). The recently discovered different populations of resting cells, in particular quiescent (Q) and non-quiescent (NQ) cells, were analyzed separately regarding their development of mutations. Quiescence is a poorly understood state of the cell cycle. Q cells are dense, unbudded daughter cells in contrast to NQ cells, which war less dense, heterogeneous and become rather apoptotic. S. cerevisiae cells in glucose-limited culture become Q and NQ cells and due to their different physical properties they can be isolated using density gradient centrifugation. The isolation is the basis for further experiments regarding oxidative stress levels and their mutation frequencies. The experiments were carried out with different yeast strains. I analyzed the wildtype (concerning DNA repair mechanisms) strain YLBM/EH150 and strains with imbalanced or knocked out DNA repair pathways YLB4, YLB14, YLBA1, and YLB14A1. For the detection of oxidative stress via microscope and FACS I used MS for the detection of superoxide anion. Furthermore, the mutations frequencies were determined using the canavanine assay, which is a forward mutation assay. In the course of this assay I determined the number of mutations in a gene that is independent of the selective pressure. In addition the differences of DNA Double strand breaks (DSBs) occurring during glucose starvation in Q and NQ cells were analyzed via Western Blot. All these experiments were needed to reveal undiscovered properties and behaviour of stationary phase cells. The understanding of the accumulation of mutations in resting cells is important, because they are a potential risk for cancer development.Die Stabilität des Genoms ist essentiell für die Gesundheit des Individuums. Mutationen entstehen nicht nur in replizierenden Zellen, sondern auch in ruhenden Zellen und können Krebs und Alterungsprozesse verursachen. Potentieller Auslöser für Mutationen sind DNA Läsionen. Ein großer Teil der menschlichen somatischen Zellen befindet sich im nicht replizierenden Zustand. Daher ist es von entscheidender Bedeutung, die Mechanismen in ruhenden Zellen kennenzulernen, die solchen Läsionen, bzw. den entstehenden Mutationen, zugrundeliegen. Im Rahmen der Diplomarbeit wurden die verschiedenen Aspekte der Mutationsentstehung in replizierenden und nicht replizierenden Zellen von Saccharomyces cerevisiae (S. cerevisiae) untersucht wurden. Die erst kürzlich entdeckten unterschiedlichen Populationen ruhender Zellen - „eigentlich ruhende“, sogenannte „Quieszente Zellen“ (Q), und eine heterogenere Gruppe, die eher apoptotisch werdenden „Nicht Quieszenten Zellen“ (NQ) - wurden getrennt voneinander untersucht. Q Zellen sind im Gegensatz zu NQ Zellen dichtere Tochterzellen, während NQ Zellen weniger dicht, heterogen und eher apoptotisch sind. S. cerevisiae Zellen in Kultur mit limitierten Nährstoffen entwickeln sich zu Q und NQ Zellen und können durch Dichtegradientenzentrifugation isoliert werden. Die Isolierung ist die Basis für weitere Experimente hinsichtlich des oxidativen Stresses sowie der Mutationsfrequenzen. Die Experimente wurden über einen Zeitverlauf mit unterschiedlichen Hefestämmen durchgeführt. Es wurden folgende Stämme untersucht: Wildtyp (hinsichtlich DNA Reparatur Mechanismen) YLBM/EH150, sowie die Stämme YLB4, YLB14, YLBA1 und YLB14A1, bei denen DNA Reparaturwege beinträchtigt oder völlig ausgeschalten sind. Oxidativer Stress wurde unter Verwendung des Fluoreszenzfarbstoffes MitoSox Red mittels FACS detektiert. Die Mutationsfrequenzen wurden durch einen „forward mutation assay“ (canavanine assay) bestimmt. Zusätzlich wurden die Unterschiede an DNA Schäden, die im Verlauf der Glukose-Hungerung auftreten, durch Western Blot untersucht. Diese Experimente sollen zur Aufklärung der Mechanismen der Mutationsentstehung in ruhenden Zellen beigetragen. Diese sind insofern von Bedeutung, als sie ein Risiko für die Entstehung von Krebs darstellen

First dedicated observations of runaway electrons in the COMPASS tokamak

Runaway electrons present an important part of the present efforts in nuclear fusion research with respect to the potential damage of the in-vessel components. The COMPASS tokamak a suitable tool for the studies of runaway electrons, due to its relatively low vacuum safety constraints, high experimental flexibility and the possibility of reaching the H-mode D-shaped plasmas. In this work, results from the first experimental COMPASS campaign dedicated to runaway electrons are presented and discussed in preliminary way. In particular, the first observation of synchrotron radiation and rather interesting raw magnetic data are shown

Formation of convective cells in the scrape-off layer of the CASTOR tokamak

Understanding of the scrape-off layer (SOL) physics in tokamaks requires diagnostics with sufficient temporal and spatial resolution. This contribution describes results of experiments performed in the SOL of the CASTOR tokamak (R=40 cm, a = 6 cm) by means of a ring of 124 Langmuir probes surrounding the whole poloidal cross section. The individual probes measure either the ion saturation current of the floating potential with the spatial resolution up to 3 mm. Experiments are performed in a particular magnetic configuration, characterized by a long parallel connection length in the SOL, L_par ~q2piR. We report on measurements in discharges, where the edge electric field is modified by inserting a biased electrode into the edge plasma. In particular, a complex picture is observed, if the biased electrode is located inside the SOL. The poloidal distribution of the floating potential appears to be strongly non-uniform at biasing. The peaks of potential are observed at particular poloidal angles. This is interpreted as formation of a biased flux tube, which emanates from the electrode along the magnetic field lines and snakes q times around the torus. The resulting electric field in the SOL is 2-dimensional, having the radial as well as the poloidal component. It is demonstrated that the poloidal electric field E_pol convects the edge plasma radially due to the E_pol x B_T drift either inward or outward depending on its sign. The convective particle flux is by two orders of magnitude larger than the fluctuation-induced one and consequently dominates.Comment: 12th International Congress on Plasma Physics, 25-29 October 2004, Nice (France